The emergent human pathogen Vibrio parahaemolyticus is a major cause of gastroenteritis worldwide. Although some virulence mechanisms have been identified, these do not fully explain the ability of this pathogen to cause disease. In this study we will elucidate mechanisms that contribute to colonization and virulence by a novel comparative approach. A common feature of human pathogenic Vibrios, including V. parahaemolyticus, is that many of their virulence factors are horizontally acquired, and yet acquired virulence traits must be coordinately regulated along with other colonization traits. Exhaustively targeting all unique sequences would be inefficient, and would provide little insight into regulatory networks that connect virulence and colonization. We propose that a comparison of colonization between this pathogen and a related bacterium that is incapable of causing disease, such as the well characterized light organ symbiont Vibrio fischeri, will indicate which mechanisms of colonization are ancient and conserved in the genera. More importantly, the novel comparison will reveal uniquely regulated transcripts that contribute specifically to the pathogenic lifestyle of V. parahaemolyticus. We hypothesize that GacA-regulated genes in V. parahaemolyticus that are not GacA-regulated or absent in V. fischeri contribute to the virulence and colonization by V. parahaemolyticus. We base our hypothesis and predictions on the fact that 1) horizontally acquired virulence factors in pathogenic Vibrios including cholera toxin are controlled by regulators conserved throughout the genus including the symbiotic bacterium, Vibrio fischeri, 2) GacA regulates symbiosis factors and host-colonization in V. fischeri and virulence and colonization in many pathogens including Vibrio cholerae, 3) key symbiotic factors that are not conserved in all Vibrios (lux) are regulated by GacA in V. fischeri indicating lifestyle-specific traits map to this regulon. The specific aims of the study are to: * Generate a global colonization-defective strain of V. parahaemolyticus through a mutation in GacA. * Use comparative DNA array analysis of GacA regulons in V. fischeri and V. parahaemolyticus to identify putative colonization and virulence traits. * Determine the role of targeted GacA-regulated genes in cytotoxicity. PUBLIC HEALTH RELEVANCE: The last decade marked the emergence of a pandemic strain of V. parahaemolyticus, which caused outbreaks in regions that were previously not considered to be at risk, foreshadowing the increasing threat to human health due to climate changes. A major obstacle to assessing risk of infection and developing preventative protocols is a lack of understanding of the pathogenicity of V. parahaemolyticus. Our findings will provide invaluable insight into the virulence mechanisms of this important emergent pathogen revealing host vulnerabilities and providing new opportunities to prevent and treat infections.